Engineering of halophilic enzymes: two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases

Abstract

Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy-terminal region of HaNDK are Glu-Glu, while those of its homologous counterpart of non-halophilic Pseudomonas NDK (PaNDK) are Ala-Ala. The double mutation, E134A-E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E-A135E in PaNDK confers halophilic characters to this enzyme, indicating that the charged state of these two residues that are located in the C-terminal region plays a critical role in determining halophilic characteristics. The importance of these two residues versus the net negative charges will be discussed in relation to the halophilicity of NDK.

Figure 1.

SDS-PAGE analysis of wild-type and mutant NDKs. MW, molecular weight marker; Ha, HaNDK/EE wild type; Pa, PaNDK/AA wild type; HaAA, HaNDK/AA mutant; PaEE, PaNDK/EE mutant.

Figure 2.

NaCl concentration dependence of enzymatic activity. Concentration of KCl in the reaction mixture was 50 mM in this experiment. (A) HaNDK/EE (Ha) and its mutant HaNDK/AA (HaAA) are shown. (B) PaNDK/AA (Pa) and its mutant PaNDK/EE (PaEE) are shown. Vertical bars show the standard deviation of six measurements.

Figure 3.

Refolding efficiency of NDK proteins from heat denaturation. NDK proteins at protein concentrations of 0.25, 0.4, 0.8, and 1.0 mg/mL were heat-treated at 85°C for 5 min, cooled, kept on ice for 30 min, and then measured for enzymatic activity at 30°C. (A) Gray and black bars represent the HaNDK/EE wild type and HaNDK/AA mutant, respectively. (B) Gray and black bars represent the PaNDK/AA wild type and PaNDK/EE mutant, respectively. All of the experiments were carried out more than three times, and average values are shown.

Figure 4.

Effects of 0.4 M NaCl on the dilution-induced inactivation of NDKs. NDKs were incubated at 30°C for the time period (6–60 min) shown in the figure, in the presence (black bars) or absence (gray bars) of 0.4 M NaCl. Enzymatic activity immediately after dilution was taken as 100%. Lane 1, HaNDK/EE (Ha); lane 2, PaNDK/EE (PaEE); lanes 3 and 5, PaNDK/AA (Pa); lanes 4 and 6, HaNDK/AA (HaAA). Average values of more than three experiments are shown.

Figure 5.

Circular dichroism profile of NDK proteins. (A) Far-UV CD spectra of wild-type NDKs are shown. Ha and Pa represent HaNDK/EE and PaNDK/AA, respectively. (B) CD₂₁₀/CD₂₂₁ ratios are shown. White bars represent wild-type HaNDK/EE (Ha) and PaNDK/AA (Pa). Dotted bars represent the mutant-type NDKs, HaNDK/AA (HaAA) and PaNDK/EE (PaEE). As the protein concentration was determined by the Lowry method, the mean residue ellipticity (Mol. Ellip.) is subject to uncertainty due to possible errors in the protein concentration.

Figure 6.

Thermal melting of NDK proteins. Thermal melting at a protein concentration of 0.5 mg/mL was followed at 220 nm. (A) HaNDK/EE (Ha) and its mutant HaNDK/AA (HaAA). (B) PaNDK/AA (Pa) and its mutant PaNDK/EE (PaEE). The first scan (“PaEE first scan”) was stopped at 60°C, cooled to 20°C, and rescanned from 40°C to 67°C (PaEE second scan).